Identification of gene expression elements in Histomonas meleagridis using splinkerette PCR, a variation of ligated adaptor PCR.

Abstract

Histomonas meleagridis is the causative agent of blackhead disease in gallinaceous birds. Limited genetic information exists for this organism, with the majority of sequence information coming from the coding regions of genes. No information is available for intergenic regions that contain DNA elements required for the regulation of gene expression. In this study, we demonstrate that splinkerette PCR, a variation of ligated adaptor PCR, can be used to identify regions of unknown sequence that lie upstream and downstream of known genomic sequences. Using this technique, we identified upstream sequences of 2 β-tubulin genes. Sequence analysis identified the 5' coding portions of the β-tubulin genes, the intergenic regions, and 2 different open reading frames encoding for a putative serine/threonine phosphatase and a putative ras-related protein, racG. We predict that these intergenic regions contain polyadenylation and cleavage signals for the 2 open reading frames and initiator elements for the β-tubulin genes. Our research demonstrates the use of splinkerette PCR as a valuable tool to identify unknown DNA sequences. In addition, the identification of the regulatory elements necessary for gene transcription in H. meleagridis will provide tools for future studies on its gene expression.